Vitamin D functions as a potent regulator of bone and calcium homeostasis as well as of cellular differentiation and replication in many target tissues. It acts as its dihydroxylated metabolite (1,25-dihydroxyvitamin D, or calcitriol) through the highly specific vitamin D receptor (1). This trans-acting transcriptional activator protein mediates calcitriol action in the regulation of the expression of target genes. Cloning the vitamin D receptor gene (2,3) showed it to be a member of the ligand-activated receptor superfamily that includes the receptors for steroid hormones (glucocorticoids, progesterone, estrogen, androgen, and mineralocorticoids) as well as thyroid hormones and vitamin A derivatives (4,5), natural regulators of a large number of physiological and developmental processes. The mechanisms by which these receptor proteins mediate the regulation of gene expression has been a subject of intense research. Rare overt mutations have been identified that compromise the function of receptors and that cause major functional disorders in humans and animals. For example, mutations in the vitamin D receptor gene, resulting in vitamin D-resistant rickets (6), and in the androgen receptor, resulting in androgen insensitivity (7), have been reported, and in the estrogen receptor gene an infrequent natural polymorphism has been correlated with a high rate of spontaneous abortion (8). However, despite a wealth of molecular information, little is known of the potential contribution of natural allelic variation in receptor genes to diversity of response to steroidal hormones in normal physiology and in disease states.
Osteoporosis is a major public health problem among the elderly in most Western countries involving both enormous health care costs and debilitating long-term effects (Riggs NEJM). Since therapy of established osteoporosis remains far from satisfactory, prevention is the best choice. Preventative strategies for osteoporosis must focus upon development of peak bone density in early adulthood and minimisation of age-related and postmenopausal bone loss. Evidence from twin and family studies have shown strong genetic effects on peak bone density that is modifiable by hormonal factors, nutrition and life style (Kelly et al, OI). Twin studies have demonstrated that monozygotic twin pairs have a much greater concordance for axial and appendicular bone density than do dizygotic pairs. Analysis of these data indicated that these genetic factors account for approximately 75% of the total variation on bone density. This effect has been confirmed in mother-daughter pair studies. The present inventors analysed the potential mechanisms of this genetic effect in the twin model. The present inventors found that the genetic effect was apparent in certain biochemical indices of bone turnover, such as osteocalcin, a marker of bone formation. Moreover amongst dizygotic twins the higher osteocalcin level was associated with the lower bone density. The present inventors have also found that the genetic effect can be shown with equal strength in another marker of bone formation, i.e., procollagen type I C-terminal propeptide and less strongly in a marker of bone breakdown, collagen type I C-terminal telopeptide. Under normal circumstances bone formation and bone breakdown are tightly linked or "coupled" in the twin physiological process of bone turnover. Thus the somewhat surprising results from the twin studies indicate that the bone formation markers, as markers of bone turnover, predict bone density and that genetic regulation of bone turnover is the pathway of the strong genetic effect on bone density.
The cross-sectional data on bone density in twins suggested that a single gene or set of genes is responsible for the genetic effect on bone density. However, it was unknown how this effect is mediated and which gene or genes influence bone density. In recent studies, using restriction fragment length polymorphism, the present inventors have shown common allelic variation in the vitamin D receptor (VDR) locus predict osteocalcin, independent of age, sex or menopausal status (Morrison et al, PNAS). The vitamin D receptor gene, as the active hormonal form of vitamin D (1,25-dihydroxyvitamin D) is an important central regulator of bone and calcium homeostasis modulating intestinal calcium absorption, bone formation, recruitment of the bone resorbing cell (osteoclast) and bone resorption per se as well as parathyroid hormone production and vitamin D's own activation in the kidney. Because of the likelihood that any alterations in the receptor for the active hormonal form of vitamin D could have such wide effects, the effect of these common VDR gene alleles on bone density was examined using a twin model. In the twin model, within-pair comparisons eliminate age and various cohort effects as confounders.
The studies have shown that common allelic variants in the VDR gene predict differences in bone density and account for 50-75% of the total genetic determination of bone density in the spine and hip.
It is believed that this a clear example that genotypic variations in transcriptional regulators of genes encoding regulatory and/or structural proteins, determine physiological set-points and predisposition to pathophysiological states with implications for susceptibility to disease and for determining likely responses to therapy.
Accordingly in a first aspect the present invention consists in a method of assessing in an individual's predisposition to a pathophysiological state and/or likely response to therapy comprising analysing genotypic variations in transcriptional regulators of genes encoding regulatory and/or structural proteins.
In a second aspect the present invention consists in a method of predicting predisposition of an individual to low or high bone density comprising analysing allelic variation within the vitamin D receptor gene of the individual.
in a preferred embodiment of the present invention the analysis comprises restriction fragment length polymorphism using endonuclease digestion.
In a further preferred embodiment of the present invention a segment of the vitamin D receptor is amplified using polymerase chain reaction prior to endonuclease digestion.
In yet a further preferred embodiment of the present invention the endonuclease is selected from the group consisting of Bsm1, Apa1, EcoRv and Taq1, and is most preferably Bsm1.
In another preferred embodiment of the present invention the segment of the vitamin D receptor is amplified using a pair of primers selected from the group consisting of
5'-CAACCAAGACTACAAGTACCGCGTCAGTGA-3' (SEQ ID:NO2) PA1 and 5'-AACCAGCGGAAGAGGTCAAGGG-3'.lambda. (SEQ ID:NO3); PA1 and 5'-CAGAGCATGGACAGGGAGCAAG-3' (SEQ ID:NO4) PA1 and 5'-GCAACTCCTCATGGCTGAGGTCTCA-3' .lambda. (SEQ ID:NO5). PA1 5'-CAACCAAGACTACAAGTACCGCGTCAGTGA-3' and PA1 5'-AACCAGCGGAAGAGGTCAAGGG-3',or PA1 5'-CAGAGCATGGACAGGGAGCAAG-3' and PA1 5'-GCAACTCCTCATGGCTGAGGTCTCA-3'.
In a second aspect the present invention consists in a primer pair derived from the sequence of the VDR gene shown in Table 5 for use in amplifying a segment of the VDR gene using polymerase chain reaction, the segment including at least one of the Bsm1, Apa1 or Taq1 cut sites as shown in Table 5.
In a preferred embodiment of this aspect of the present invention the primer pair is
The allelic makeup of other transacting factors which may be assessed include oestrogen and androgen receptors to determine risk of osteoporosis and/or ischaemic heart disease. The allelic makeup of the androgen receptor may be also used to assess risk and responsiveness to therapeutic intervention in skin diseases. The allelic makeup of the glucocorticoid receptor and the retinoic acid receptor can be determined to assess risk of osteoporosis. The allelic makeup of the mineralocorticoid receptor can be determined to assess risk of hypertension and the allelic makeup of proto-oncogenes can be determined to assess cancer risk. Tissue specific regulators can also be assessed to determine osteoporosis/cancer risk.